CN101851637B

CN101851637B - Method for preparing transgenic animal for expressing multiple genes simultaneously

Info

Publication number: CN101851637B
Application number: CN2010100343183A
Authority: CN
Inventors: 李奎; 鞠辉明; 樊俊华; 白立景; 牟玉莲; 杨述林; 唐中林; 崔文涛
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2010-01-15
Filing date: 2010-01-15
Publication date: 2011-12-14
Anticipated expiration: 2030-01-15
Also published as: CN101851637A; US20120233717A1; US8742085B2; WO2011085528A1

Abstract

The invention discloses a method for preparing a transgenic animal for expressing a plurality of genes simultaneously. The invention also discloses a method for preparing a transgenic embryo. In the method for preparing the transgenic embryo, the transgenic embryo is obtained by transferring both a phytase gene and a human myxovirus resistance gene A into a target embryo. The transgenic animal for expressing the plurality of the genes can be obtained by transplanting the transgenic embryo into a female target animal. The method has the remarkable advantage that the plurality of the genes are expressed simultaneously by primary transgenosis and provides a convenient means for preparing a transferred united gene animal and the like.

Description

The method of the transgenic animal of a plurality of genes is expressed in a kind of preparation simultaneously

Technical field

The present invention relates to biological technical field, the method for the transgenic animal of a plurality of genes is expressed in particularly a kind of preparation simultaneously.

Background technology

In the research of traditional preparation process transgenic animal, often realize by carrier for expression of eukaryon, by molecular biology method target gene fragment is connected to the eukaryotic promoter downstream, can instruct exogenous gene expression after being incorporated in the cellular genome, this method has in the research of genetic expression and function extremely widely to be used, the each transgenosis of tradition transgenic method is only integrated a goal gene, and then the transgenic animal of a gene are expressed in preparation, if relate to preparation polygene transgenic animal, then need the corresponding single-gene transgenic animal of preparation separately, change the offspring of polygene animal then by the preparation of the methods such as mating between transgenic animal, this method for preparing the polygene animal wastes time and energy and expends height.

Summary of the invention

The object of the present invention is to provide a kind of method for preparing transgenic embryos.

The method for preparing transgenic embryos provided by the invention is that phytase gene and people myxovirus resistant gene A are all imported among the purpose embryo, obtains transgenic embryos.

Sequence 1 holds the 1360-2658 position from 5 ' in the nucleotide sequence of above-mentioned phytase gene such as the sequence table; Sequence 1 holds the 3803-5791 position from 5 ' in the nucleotide sequence of above-mentioned people myxovirus resistant gene A such as the sequence table.

Above-mentioned phytase gene and people myxovirus resistant gene A import among the embryo by the dna fragmentation of nucleotide sequence shown in sequence in the sequence table 1.

Above-mentioned embryo is a pronuclear-stage embryos.

Above-mentioned embryo specifically can be the embryo of pig, ox, sheep, cat, dog, rabbit or mouse for the embryo of any animal except that the people.

Another purpose of the present invention is to provide a kind of method of cultivating transgenic animal.

The method of cultivation transgenic animal provided by the invention is that the transgenic embryos that the above-mentioned method for preparing transgenic embryos is prepared is transplanted in the body of female purpose animal, obtains expressing simultaneously the transgenic animal of a plurality of genes.

The above-mentioned purpose animal can be any animal except that the people, specifically can be pig, ox, sheep, cat, dog, rabbit or mouse.

The carrier for expression of eukaryon pcDNA-appA-MxA of Expressing Recombinant Phytase and people myxovirus resistant gene when the present invention utilizes structure utilizes microinjection to prepare to contain the transgenic pig of dual-gene (phytase gene (appA) and people myxovirus resistant gene A (MxA)).Can detect the transgenic pig kind simultaneously and integrate dual-gene carrier significant advantage of the present invention simultaneously and show that transgenosis realizes polygenicly expressing simultaneously, changeing associating genetic animal etc. for preparation provides a kind of means easily.

Description of drawings

Fig. 1: amplification appA gel electrophoresis figure.

Fig. 2: pcDNA-appA plasmid map.

Fig. 3: amplification MxA gel electrophoresis figure.

Fig. 4: pcDNA-MxA plasmid map.

Fig. 5: pcDNA-appA-MxA plasmid map.

Fig. 6: the expression of embryo's detection by quantitative appA and MxA behind the microinjection AMP gene.

Fig. 7: AMP transgenic pig PCR detects foreign gene.

Fig. 8: AMP transgenic pig SOUTHERN BLOT detects foreign gene.

The expression of Fig. 9: detection by quantitative appA and MxA.

Embodiment

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

Among the following embodiment, if no special instructions, be ordinary method.

The preparation of embodiment 1, transgenic embryos

One, the structure of recombinant expression vector

Embodiment 1, express the structure of dual-gene carrier

1, the acquisition of single-gene expression vector basic building block

(1) preparation of carrier for expression of eukaryon pcDNA-appA is a template with bacillus coli DH 5 alpha (available from the full formula in Beijing King Company, article No. CD201), with primer amplification phytase gene (appA) fragment of the amplification appA that enumerates in the table 1.Reaction system is that reaction system is 50 μ l, 5 μ L, 10 * Buffer, 8 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer appA-L1,1 μ L, 20 μ M primer appA-R1 (primer sequence sees Table 1), 0.5 μ L5U/ μ L high-fidelity Tag polysaccharase, 2 μ l bacillus coli DH 5s

α does template, adds ultrapure water to 50 μ L (the high-fidelity enzyme is precious biological available from Dalian, article No. DR010A).The pcr amplification program: 98 ℃ of 10s, 68 ℃ of 2min circulate 30 times.Pcr amplification product detects (as shown in Figure 1) with 1% agarose gel electrophoresis.

The PCR product reclaims purifying (operation steps is seen company's test kit) with QIAGEN sepharose test kit, with HindIII, EcoR I double digestion purified product; Use HindIII, EcoR I double digestion pcDNA3.1 (+) carrier (available from invitrogen company) simultaneously; Pcr amplification product is connected with carrier after enzyme is cut (ligase enzyme available from promega company, article No. M1804); Transform DH5 α (available from the full formula in Beijing King Company, article No. CD201) competence intestinal bacteria by company's requirement operation steps; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing company); Choose sequencing result and show the carrier that contains appA (nucleotide sequence is held shown in the 1360-2658 position from 5 ' as sequence in the sequence table 1), it is standby to extract plasmid, and this order-checking is shown correct plasmid called after pcDNA-appA (plasmid map as shown in Figure 2).

(2) preparation of carrier for expression of eukaryon pcDNA-MxA

With people cDNA (extract stripped blood of the people extract total RNA, reverse transcription) is template, with primer amplification people myxovirus resistant gene A (MxA) fragment of the amplification MxA that enumerates in the table 1.Reaction system is that reaction system is 50 μ l, 5 μ L, 10 * Buffer, 8 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer Mx-L1,1 μ L, 20 μ M primer Mx-R1 (sequence sees Table 1), 0.5 μ L5U/ μ L high-fidelity Tag polysaccharase, 100ng people cDNA is a template, add ultrapure water to 50 μ L (the high-fidelity enzyme is precious biological available from Dalian, article No. DR010A).The pcr amplification program: 98 ℃ of 10s, 68 ℃ of 2min,, circulate 30 times.Pcr amplification product detects (Fig. 3) with 1% agarose gel electrophoresis.

The PCR product reclaims purifying (operation steps is seen company's test kit) with QIAGEN sepharose test kit, with HindIII, EcoR I double digestion purified product; Use HindIII, EcoR I double digestion pcDNA3.1 (+) carrier (available from invitrogen company) simultaneously; Pcr amplification product is connected with carrier after enzyme is cut (ligase enzyme available from promega company, article No. M1804); Transform DH5 α (available from the full formula in Beijing King Company, article No. CD201) competence intestinal bacteria by company's requirement operation steps; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators; Choose single colony inoculation and cultivate in liquid LB substratum, part bacterium liquid send company's order-checking (invitrogen Beijing company); Choose sequencing result and show the carrier that contains MxA fragment (nucleotide sequence is held shown in the 3803-5791 position from 5 ' as sequence in the sequence table 1), the extraction plasmid is standby, and this order-checking is shown correct plasmid called after pcDNA-MxA (plasmid map as shown in Figure 4).

2, double gene expression vector AMP makes up

With above-mentioned pcDNA-appA (plasmid map as shown in Figure 2) is template, with the listed listed respective members of primer amplification table 1 of table 1.5 μ L, 10 * Buffer, 8 μ L 2.5mM dNTP, 1 μ L, 20 μ M primer CMV-appA-L1,1 μ L, 20 μ M primer CMVappA-R1 (sequence sees Table 1), 0.5 μ L5U/ μ L high-fidelity Tag polysaccharase, 100ng pcDNA-appA plasmid, add ultrapure water to 50 μ L (the high-fidelity enzyme is precious biological available from Dalian, article No. DR010A).The pcr amplification program: 98 ℃ of 10s, 68 ℃ of 3min circulate 30 times.Pcr amplification product detects with 1% agarose gel electrophoresis.PCR product partly check order (invitrogen company); Showing the CMV-appA-BGH pA fragment that amplification obtains (nucleotide sequence as sequence in the sequence table 1 from 5 ' end 618-2977 position shown in); CMV-appA-BGH pA fragment is by CMV promotor, appA and the BGH terminator of polyphone are formed successively; Wherein the CMV promotor (nucleotide sequence as sequence in the sequence table 1 from 5 ' end 671-1358 position shown in), appA (nucleotide sequence as sequence in the sequence table 1 from shown in 5 ' end 1360-2658 position) and BGH terminator (nucleotide sequence is shown in 5 of sequence in the sequence table 1 ' end 2735-2959 position).

Above-mentioned PCR product C MV-appA-BGH pA fragment reclaims purifying (operation steps is seen company's test kit) with QIAGEN sepharose test kit, cuts purified product with Mlu I enzyme; Use Mlu I single endonuclease digestion pcDNA-MxA plasmid simultaneously; Pcr amplification product is connected with carrier after enzyme is cut (ligase enzyme available from promega company, article No. M1804); Transform DH5 α (available from the full formula in Beijing King Company, article No. CD201) competence intestinal bacteria by company's requirement operation steps; Bacterium liquid is coated with agarose gel plate, overnight incubation in 37 ℃ of incubators.

Choosing single colony inoculation cultivates in liquid LB substratum, bacterium liquid PCR detects and inserts (the same amplification of the amplification system CMV-appA-BGH pA fragment part (primer sees Table 1) of CMV-appA-BGH pA fragment in the fragment, wherein template changes bacterium liquid 2 microlitres into), the result has corresponding CMV-appA-BGH pA fragment amplification band, send company's order-checking (invitrogen Beijing company) with the part bacterium liquid of above-mentioned difference; The result shows the recombinant expression vector that obtains to contain CMV-appA-BGH pA fragment and MxA expression cassette, with this carrier called after pcDNA-appA-MxA, abbreviates AMP as.The plasmid map of AMP as shown in Figure 5.

Order-checking shows that AMP has the nucleotide sequence of sequence 1 in the sequence table, contains appA expression cassette and MxA expression cassette in its sequence.

Wherein the appA expression cassette is made up of placed in-line CMV promotor successively (nucleotide sequence as sequence in the sequence table 1 from shown in 5 ' end 671-1358 position), appA (nucleotide sequence as sequence in the sequence table 1 from shown in 5 ' end 1360-2658 position) and BGH terminator (nucleotide sequence is shown in 5 of sequence in the sequence table 1 ' end 2735-2959 position); The MxA expression cassette is by placed in-line CMV promotor (nucleotide sequence is shown in 5 of sequence in the sequence table 1 ' end 3114-3802 position), MxA (nucleotide sequence is shown in 5 of sequence in the sequence table 1 ' end 3803-5791 position) and BGH terminator (nucleotide sequence is shown in 5 of sequence in the sequence table 1 ' end 5868-6092 position) are formed successively.

Two, the preparation of transgenic embryos

1, the pcDNA-appA-MxA that step 1 is obtained uses ScaI (available from fermentas company, article No. ER0431) and SmaI (available from fermentas company, article No. ER0661) band of agarose gel electrophoresis recovery 6.9Kb size behind the double digestion is diluted to 5ng/ μ l.Order-checking shows that the nucleotide sequence of the band of this 6.9Kb size is shown in sequence in the sequence table 1.

Utilize the method for microinjection to be expelled in the pronuclear-stage embryos of pig the linearizing fragment of above-mentioned 6.9Kb size, obtain transgenic embryos, (available from millipore company, article No. MR-182-D) is cultured to 8 cell stages in NCSU 23 substratum.

2, transgenic embryos detects

The genomic dna of getting part embryo wherein is directly as template, according to ProtoScript M-MuLV TaqRT-PCR test kit (available from NEB company, article No. E6400S) operation instructions (primer is the quantitative PCR detection primer of cited appA of table 1 and MxA), (primer is the appA-RTL1 in the table 1 directly to detect appA among the embryo

And appA-RTR1) and MxA (primer is hMx1-RTL1 and the hMx1-RTR1 in the table 1).The PCR reaction system is 50 μ l, 5 μ L10 * Buffer, 8 μ L2.5mM dNTP, 1 μ L20 μ M upstream primer, 1 μ L20 μ M downstream primer, 0.5 μ L5U/ μ L high-fidelity Tag polysaccharase, add ultrapure water to 50 μ L, piggy genomic dna with acquisition is a template to be detected, pcr amplification program: 95 ℃ of 5min; 94 ℃ of 20s, 56 ℃ of annealing temperatures, 72 ℃ of 1m circulate 30 times, and last 72 ℃ are extended 5min.Pcr amplification product detects with 1.0% agarose gel electrophoresis, filters out and can amplify appA, MxA gene respectively, and the result shows that among 24 embryos that detected, the 13rd and No. 17 embryo detects and can express appA gene and MxA gene (Fig. 6) simultaneously.

The preparation of embodiment 2, transgenic animal

One, preparation transgenic animal

Press the transgenic embryos of method preparation among the embodiment 1, ectogenesis will obtain 38 transgenic pigs through cultivating at the external sow horn of uterus of oestrusing synchronous of being transplanted to by the vagina uterine neck then to 8 cell stages.

Two, the integration of transgenic pig detects

1, PCR detects

The integration that utilizes appA and MxA detection by quantitative primer (seeing Table 1) to detect corresponding gene in the transgenic pig genomic dna detects the integration situation of foreign gene in the above-mentioned transgenic pig.

PCR reaction system and amplification system detect identical with the transgenic embryos of the step 2 of embodiment 1, filter out the pig (Fig. 7) that can amplify appA and MxA gene respectively (among the figure ,+: positive control, pcDNA-appA-Mx plasmid are template;-: negative control, normal pig DNA is a template; 1-23: be different transgenic progeny pig amplified bands).

From the last figure of Fig. 7 as seen: 3,6,11,17,20, No. 22 pig genomic dnas can amplify the band about 355bp in the 1-23 pig, and band is consistent with the appA band of expection amplification; 3,6,11,17,20, No. 22 pig genomic dnas can expand the 325bp band and expect that the MxA band of amplification is consistent in the pig from the visible 1-23 pig of figure below of Fig. 7.The amplified production order-checking shows that the segmental nucleotide sequence that amplifies is all correct.

2, southen hybridization detects

Quantitative PCR detection primer with appA in the table 1 and MxA is a primer respectively, according to test kit process specifications system application of sample; The PCR working procedure is as follows: 95 ℃ of 5min, and 94 ℃ of 45S, 56 ℃ of 1min, 72 ℃ of 1min, 30 circulations are extended 10min (the probe mark test kit are available from innogen-cn company, article No.: DDLK-010), obtain two PCR products for back 72 ℃.

Be that probe carries out southen hybridization and detects with above-mentioned two PCR products respectively, before the hybridization 95 ℃ of sex change place in the frozen water immediately after 5 minutes 10 minutes standby; The pig ear tissue DNA that is numbered 3,6,11,17,20, No. 22 transgenic pigs that identified in a large amount of extraction steps 1 with HindIII and Not I double digestion genome, concentrates the genome enzyme and cuts product to 300ng/ μ l; Separate each endonuclease bamhi in 1% agarose gel electrophoresis, sample 50 microlitres on every hole; Transfer on the positively charged nylon membrane after making the sex change of DNA original position then.Carry out prehybridization, hybridization and X-mating plate exposure tests hybridization signal (available from innogen-cn company, article No.: DIGD-210) according to the test kit operation instructions.Detected result (Fig. 8) shows that appA gene and MxA gene have been integrated in being numbered in 3,6,11,17,20, No. 22 transgenic pigs that southern hybridization detects.

Three, RT-PCR detects

The pig ear vein that is numbered 3,6,11,17,20, No. 22 transgenic pigs that sifts out from step 2 is respectively gathered 100 microliters of blood, according to sky root blood total RNA extraction reagent box (article No.: DP433) operation instructions, extract and respectively to organize cell total rna; According to TOYOBO reverse transcription test kit (article No.: FSK-100), total RNA reverse transcription of extracting is become cDNA.

Be template with above-mentioned cDNA respectively, with appA in the table 1 and MxA quantitative PCR detection primer, carry out sxemiquantitative PCR and detect appA and MxA, be confidential reference items (primer sees Table 1) wherein with pig GAPDH gene, the PCR reaction system is 50 μ l, 5 μ L10 * Buffer, 8 μ L 2.5mM dNTP, 1 μ L, 20 μ M upstream primers, 1 μ L, 20 μ M downstream primers (primer of amplification appA, MxA and pig GAPDH sees Table 1 respectively), 0.5 μ L5U/ μ L high-fidelity Tag polysaccharase, the 100ng template adds ultrapure water to 50 μ L.

Pcr amplification program: 95 ℃ of 5min; 94 ℃ of 20s, 56 ℃ of annealing temperatures, 72 ℃ of 1m,, last 72 ℃ are extended 5min, 23 circulations during confidential reference items GAPDH amplification, 32 circulations of other template amplifications.

Pcr amplification product detects (Fig. 9) with 1.2% agarose gel electrophoresis.Semi-quantitative results shows that 3, No. 20 pigs all can detect the expression of MxA and appA, illustrates that the transgenic pig of correspondence can be expressed MxA and appA gene simultaneously.

Table 1: the primer table 1 that is used for the amplification vector member among the present invention: the present invention is used for the primer of amplification vector member

Sequence table

＜110〉Institute of Animal Sciences, Chinese Academy of Agricultural Sciences

＜120〉method of the transgenic animal of a plurality of genes is expressed in a kind of preparation simultaneously

<160>1

<210>1

<211>6915

<212>DNA

<213>1

<400>1

actcaaccaa?gtcattctga?gaatagtgta?tgcggcgacc?gagttgctct?tgcccggcgt 60

caatacggga?taataccgcg?ccacatagca?gaactttaaa?agtgctcatc?attggaaaac 120

gttcttcggg?gcgaaaactc?tcaaggatct?taccgctgtt?gagatccagt?tcgatgtaac 180

ccactcgtgc?acccaactga?tcttcagcat?cttttacttt?caccagcgtt?tctgggtgag 240

caaaaacagg?aaggcaaaat?gccgcaaaaa?agggaataag?ggcgacacgg?aaatgttgaa 300

tactcatact?cttccttttt?caatattatt?gaagcattta?tcagggttat?tgtctcatga 360

gcggatacat?atttgaatgt?atttagaaaa?ataaacaaat?aggggttccg?cgcacatttc 420

cccgaaaagt?gccacctgac?gtcgacggat?cgggagatct?cccgatcccc?tatggtgcac 480

tctcagtaca?atctgctctg?atgccgcata?gttaagccag?tatctgctcc?ctgcttgtgt 540

gttggaggtc?gctgagtagt?gcgcgagcaa?aatttaagct?acaacaaggc?aaggcttgac 600

cgacaattgc?atgaagaatc?tgcttagggt?taggcgtttt?gcgctgcttc?gcgatgtacg 660

ggccagatat?acgcgttgac?attgattatt?gactagttat?taatagtaat?caattacggg 720

gtcattagtt?catagcccat?atatggagtt?ccgcgttaca?taacttacgg?taaatggccc 780

gcctggctga?ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccat 840

agtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc 900

ccacttggca?gtacatcaag?tgtatcatat?gccaagtacg?ccccctattg?acgtcaatga 960

cggtaaatgg?cccgcctggc?attatgccca?gtacatgacc?ttatgggact?ttcctacttg 1020

gcagtacatc?tacgtattag?tcatcgctat?taccatggtg?atgcggtttt?ggcagtacat 1080

caatgggcgt?ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgt 1140

caatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc?gtaacaactc 1200

cgccccattg?acgcaaatgg?gcggtaggcg?tgtacggtgg?gaggtctata?taagcagagc 1260

tctctggcta?actagagaac?ccactgctta?ctggcttatc?gaaattaata?cgactcacta 1320

tagggagacc?caagctggct?agcgtttaaa?cttaagctta?tgaaagcgat?cttaatccca 1380

tttttatctc?ttctgattcc?gttaaccccg?caatctgcat?tcgctcagag?tgagccggag 1440

ctgaagctgg?aaagtgtggt?gattgtcagt?cgtcatggtg?tgcgtgctcc?aaccaaggcc 1500

acgcaactga?tgcaggatgt?caccccagac?gcatggccaa?cctggccggt?aaaactgggt 1560

tggctgacac?cgcgcggtgg?tgagctaatc?gcctatctcg?gacattacca?acgccagcgt 1620

ctggtagccg?acggattgct?ggcgaaaaag?ggctgcccgc?agtctggtca?ggtcgcgatt 1680

attgctgatg?tcgacgagcg?tacccgtaaa?acaggcgaag?ccttcgccgc?cgggctggca 1740

cctgactgtg?caataaccgt?acatacccag?gcagatacgt?ccagtcccga?tccgttattt 1800

aatcctctaa?aaactggcgt?ttgccaactg?gataacgcga?acgtgactga?cgcgatcctc 1860

agcagggcag?gagggtcaat?tgctgacttt?accgggcatc?ggcaaacggc?gtttcgcgaa 1920

ctggaacggg?tgcttaattt?tccgcaatca?aacttgtgcc?ttaaacgtga?gaaacaggac 1980

gaaagctgtt?cattaacgca?ggcattacca?tcggaactca?aggtgagcgc?cgacaatgtc 2040

tcattaaccg?gtgcggtaag?cctcgcatca?atgctgacga?agatatttct?cctgcaacaa 2100

gcacagggaa?tgccggagcc?ggggtgggga?aggatcaccg?attcacacca?gtggaacacc 2160

ttgctaagtt?tgcataacgc?gcaattttat?ttgttacaac?gcacgccaga?ggttgcccgc 2220

agccgcgcca?ccccgttatt?agatttgatc?aagacagcgt?tgacgcccca?tccaccgcaa 2280

aaacaggcgt?atggtgtgac?attacccact?tcagtgctgt?ttatcgccgg?acacgatact 2340

aatctggcaa?atctcggcgg?cgcactggag?ctcaactgga?cgcttcccgg?tcagccggat 2400

aacacgccgc?caggtggtga?actggtgttt?gaacgctggc?gtcggctaag?cgataacagc 2460

cagtggattc?aggtttcgct?ggtcttccag?actttacagc?agatgcgtga?taaaacgccg 2520

ctgtcattaa?atacgccgcc?cggagaggtg?aaactgaccc?tggcaggatg?tgaagagcga 2580

aatgcgcagg?gcatgtgttc?gttggcaggt?tttacgcaaa?tcgtgaatga?agcacgcata 2640

ccggcgtgca?gtttgtaaga?attctgcaga?tatccagcac?agtggcggcc?gctcgagtct 2700

agagggcccg?tttaaacccg?ctgatcagcc?tcgactgtgc?cttctagttg?ccagccatct 2760

gttgtttgcc?cctcccccgt?gccttccttg?accctggaag?gtgccactcc?cactgtcctt 2820

tcctaataaa?atgaggaaat?tgcatcgcat?tgtctgagta?ggtgtcattc?tattctgggg 2880

ggtggggtgg?ggcaggacag?caagggggag?gattgggaag?acaatagcag?gcatgctggg 2940

gatgcggtgg?gctctatggc?ttctgaggcg?gaaagaacca?gctggggctc?tagggggtat 3000

ccccacgcgc?cctgtagcgg?cgcattaagc?gcggcgggtg?tggtggttac?gcgcagcgtg 3060

accgctacac?ttgccagcgc?cctagcgccc?gctcctttcg?ctttcttccc?ttcacgcgtt 3120

gacattgatt?attgactagt?tattaatagt?aatcaattac?ggggtcatta?gttcatagcc 3180

catatatgga?gttccgcgtt?acataactta?cggtaaatgg?cccgcctggc?tgaccgccca 3240

acgacccccg?cccattgacg?tcaataatga?cgtatgttcc?catagtaacg?ccaataggga 3300

ctttccattg?acgtcaatgg?gtggagtatt?tacggtaaac?tgcccacttg?gcagtacatc 3360

aagtgtatca?tatgccaagt?acgcccccta?ttgacgtcaa?tgacggtaaa?tggcccgcct 3420

ggcattatgc?ccagtacatg?accttatggg?actttcctac?ttggcagtac?atctacgtat 3480

tagtcatcgc?tattaccatg?gtgatgcggt?tttggcagta?catcaatggg?cgtggatagc 3540

ggtttgactc?acggggattt?ccaagtctcc?accccattga?cgtcaatggg?agtttgtttt 3600

ggcaccaaaa?tcaacgggac?tttccaaaat?gtcgtaacaa?ctccgcccca?ttgacgcaaa 3660

tgggcggtag?gcgtgtacgg?tgggaggtct?atataagcag?agctctctgg?ctaactagag 3720

aacccactgc?ttactggctt?atcgaaatta?atacgactca?ctatagggag?acccaagctg 3780

gctagcgttt?aaacttaagc?ttatggttgt?ttccgaagtg?gacatcgcaa?aagctgatcc 3840

agctgctgca?tcccaccctc?tattactgaa?tggagatgct?actgtggccc?agaaaaatcc 3900

aggctcggtg?gctgagaaca?acctgtgcag?ccagtatgag?gagaaggtgc?gcccctgcat 3960

cgacctcatt?gactccctgc?gggctctagg?tgtggagcag?gacctggccc?tgccagccat 4020

cgccgtcatc?ggggaccaga?gctcgggcaa?gagctccgtg?ttggaggcac?tgtcaggagt 4080

tgcccttccc?agaggcagcg?ggatcgtgac?cagatgcccg?ctggtgctga?aactgaagaa 4140

acttgtgaac?gaagataagt?ggagaggcaa?ggtcagttac?caggactacg?agattgagat 4200

ttcggatgct?tcagaggtag?aaaaggaaat?taataaagcc?cagaatgcca?tcgccgggga 4260

aggaatggga?atcagtcatg?agctaatcac?cctggagatc?agctcccgag?atgtcccgga 4320

tctgactcta?atagaccttc?ctggcataac?cagagtggct?gtgggcaatc?agcctgctga 4380

cattgggtat?aagatcaaga?cactcatcaa?gaagtacatc?cagaggcagg?agacaatcag 4440

cctggtggtg?gtccccagta?atgtggacat?tgccaccaca?gaggctctca?gcatggccca 4500

ggaggtggac?cccgagggag?acaggaccat?cggaatcttg?acgaagcctg?atctggtgga 4560

caaaggaact?gaagacaagg?ttgtggacgt?ggtgcggaac?ctcgtgttcc?acctgaagaa 4620

gggttacatg?attgtcaagt?gccggggcca?gcaggagatc?caggaccagc?tgagcctgtc 4680

cgaagccctg?cagagagaga?agatcttctt?tgagaaccac?ccatatttca?gggatctgct 4740

ggaggaagga?aaggccacgg?ttccctgcct?ggcagaaaaa?cttaccagcg?agctcatcac 4800

acatatctgt?aaatctctgc?ccctgttaga?aaatcaaatc?aaggagactc?accagagaat 4860

aacagaggag?ctacaaaagt?atggtgtcga?cataccggaa?gacgaaaatg?aaaaaatgtt 4920

cttcctgata?gataaaatta?atgcctttaa?tcaggacatc?actgctctca?tgcaaggaga 4980

ggaaactgta?ggggaggaag?acattcggct?gtttaccaga?ctccgacacg?agttccacaa 5040

atggagtaca?ataattgaaa?acaattttca?agaaggccat?aaaattttga?gtagaaaaat 5100

ccagaaattt?gaaaatcagt?atcgtggtag?agagctgcca?ggctttgtga?attacaggac 5160

atttgagaca?atcgtgaaac?agcaaatcaa?ggcactggaa?gagccggctg?tggatatgct 5220

acacaccgtg?acggatatgg?tccggcttgc?tttcacagat?gtttcgataa?aaaattttga 5280

agagtttttt?aacctccaca?gaaccgccaa?gtccaaaatt?gaagacatta?gagcagaaca 5340

agagagagaa?ggtgagaagc?tgatccgcct?ccacttccag?atggaacaga?ttgtctactg 5400

ccaggaccag?gtatacaggg?gtgcattgca?gaaggtcaga?gagaaggagc?tggaagaaga 5460

aaagaagaag?aaatcctggg?attttggggc?tttccaatcc?agctcggcaa?cagactcttc 5520

catggaggag?atctttcagc?acctgatggc?ctatcaccag?gaggccagca?agcgcatctc 5580

cagccacatc?cctttgatca?tccagttctt?catgctccag?acgtacggcc?agcagcttca 5640

gaaggccatg?ctgcagctcc?tgcaggacaa?ggacacctac?agctggctcc?tgaaggagcg 5700

gagcgacacc?agcgacaagc?ggaagttcct?gaaggagcgg?cttgcacggc?tgacgcaggc 5760

tcggcgccgg?cttgcccagt?tccccggtta?ggaattctgc?agatatccag?cacagtggcg 5820

gccgctcgag?tctagagggc?ccgtttaaac?ccgctgatca?gcctcgactg?tgccttctag 5880

ttgccagcca?tctgttgttt?gcccctcccc?cgtgccttcc?ttgaccctgg?aaggtgccac 5940

tcccactgtc?ctttcctaat?aaaatgagga?aattgcatcg?cattgtctga?gtaggtgtca 6000

ttctattctg?gggggtgggg?tggggcagga?cagcaagggg?gaggattggg?aagacaatag 6060

caggcatgct?ggggatgcgg?tgggctctat?ggcttctgag?gcggaaagaa?ccagctgggg 6120

ctctaggggg?tatccccacg?cgccctgtag?cggcgcatta?agcgcggcgg?gtgtggtggt 6180

tacgcgcagc?gtgaccgcta?cacttgccag?cgccctagcg?cccgctcctt?tcgctttctt 6240

cccttccttt?ctcgccacgt?tcgccggctt?tccccgtcaa?gctctaaatc?gggggctccc 6300

tttagggttc?cgatttagtg?ctttacggca?cctcgacccc?aaaaaacttg?attagggtga 6360

tggttcacgt?agtgggccat?cgccctgata?gacggttttt?cgccctttga?cgttggagtc 6420

cacgttcttt?aatagtggac?tcttgttcca?aactggaaca?acactcaacc?ctatctcggt 6480

ctattctttt?gatttataag?ggattttgcc?gatttcggcc?tattggttaa?aaaatgagct 6540

gatttaacaa?aaatttaacg?cgaattaatt?ctgtggaatg?tgtgtcagtt?agggtgtgga 6600

aagtccccag?gctccccagc?aggcagaagt?atgcaaagca?tgcatctcaa?ttagtcagca 6660

accaggtgtg?gaaagtcccc?aggctcccca?gcaggcagaa?gtatgcaaag?catgcatctc 6720

aattagtcag?caaccatagt?cccgccccta?actccgccca?tcccgcccct?aactccgccc 6780

agttccgccc?attctccgcc?ccatggctga?ctaatttttt?ttatttatgc?agaggccgag 6840

gccgcctctg?cctctgagct?attccagaag?tagtgaggag?gcttttttgg?aggcctaggc 6900

ttttgcaaaa?agctc 6915

Claims

1. a method for preparing transgenic embryos is that phytase gene and people myxovirus resistant gene A are all imported among the purpose embryo, obtains transgenic embryos; Described embryo is the embryo of pig, ox, sheep, cat, dog, rabbit or mouse; Sequence 1 holds the 1360-2658 position from 5 ' in the nucleotide sequence of described phytase gene such as the sequence table; Sequence 1 holds the 3803-5791 position from 5 ' in the nucleotide sequence of described people myxovirus resistant gene A such as the sequence table; Described phytase gene and people myxovirus resistant gene A import among the embryo by the dna fragmentation of nucleotide sequence shown in sequence in the sequence table 1.

2. method according to claim 1 is characterized in that: described embryo is a pronuclear-stage embryos.

3. a method of cultivating transgenic animal is that the transgenic embryos of claim 1 or 2 described method preparations is transplanted in the body of female purpose animal, obtains expressing simultaneously the transgenic animal of a plurality of genes.

4. method according to claim 3 is characterized in that: described purpose animal is pig, ox, sheep, cat, dog, rabbit or mouse.